PCR Analysis of the Distribution of Unicellular Cyanobacterial Diazotrophs in the Arabian Sea

Abstract

An oligonucleotide primer, NITRO821R, targeting the 16S rRNA gene of unicellular cyanobacterial N 2 fixers was developed based on newly derived sequences from Crocosphaera sp. strain WH 8501 and Cyanothece sp. strains WH 8902 and WH 8904 as well as several previously described sequences of Cyanothece sp. and sequences of intracellular cyanobacterial symbionts of the marine diatom Climacodium frauenfeldianum. This oligonucleotide is specific for the targeted organisms, which represent a well-defined phylogenetic lineage, and can detect as few as 50 cells in a standard PCR when it is used as a reverse primer together with the cyanobacterium-and plastid-specific forward primer CYA359F (U. Nübel, F. Garcia-Pichel, and G. Muyzer, Appl. Environ. Microbiol. 63:3327-3332, 1997). Use of this primer pair in the PCR allowed analysis of the distribution of marine unicellular cyanobacterial diazotrophs along a transect following the 67°E meridian from Victoria, Seychelles, to Muscat, Oman (0.5°S to 26°N) in the Arabian Sea. These organisms were found to be preferentially located in warm (>29°C) oligotrophic subsurface waters between 0 and 7°N, but they were also found at a station north of Oman at 26°N, 56°35E, where similar water column conditions prevailed. Slightly cooler oligotrophic waters (<29°C) did not contain these organisms or the numbers were considerably reduced, suggesting that temperature is a key factor in dictating the abundance of this unicellular cyanobacterial diazotroph lineage in marine environments.Microbial N 2 fixation has traditionally been considered to be of minor importance as a source of fixed nitrogen in oceanic environments (5). Contemporary mass balance estimates, however, have suggested that there is excess removal of combined forms of nitrogen, particularly from areas of the tropical ocean, and it has been proposed that biological N 2 fixation may account for the imbalances (6,12). This has led to a radically revised view of the quantitative importance of this process in the nitrogen cycle. Current estimates of global N 2 fixation are ϳ240 Tg of N year Ϫ1 and a marine contribution of ϳ80 Tg of N year Ϫ1 (4). Trichodesmium, a filamentous nonheterocystous cyanobacterium broadly distributed throughout tropical and subtropical oceans, has long been considered to be responsible for most of this marine N 2 fixation (see references 3 and 17 for recent reviews). However, recently, Zehr et al. (34) showed that unicellular cyanobacteria that are 3 to 10 m in diameter may make a significant contribution to oceanic N 2 fixation. Indeed, based on N 2 fixation data for the 0.2-to 10-m bacterioplankton size fraction and the concentrations of phycoerythrin-containing unicellular cyanobacteria, they estimated that this contribution might equal or exceed that of Trichodesmium.Several unicellular marine cyanobacterial diazotrophs belonging to the genera Synechococcus (15, 16) Cyanothece (21), and Crocosphaera (28) have previously been described. These organisms all have temporal separation of N 2 fixation and...

High levels of heterogeneity in diazotroph diversity and activity within a putative hotspot for marine nitrogen fixation

“…Recently, the occurrence of the γ-24774A11 clade has been found to be positively, non-linearly correlated with temperature, with maximum abundances associated with surface waters 426°C (Moisander et al, 2014). Overall, our data are consistent with the observed distributions of these organisms across a range of oceanic provinces (Capone et al, 1997;Mazard et al, 2004;Langlois et al, 2008;Moisander et al, 2010) and supports previous findings that temperature is an important determinant of diazotroph spatiotemporal dynamics.…”

Genome-wide analysis of diel gene expression in the unicellular N2-fixing cyanobacterium Crocosphaera watsonii WH 8501

“…The fixation of N 2 is a particularly important source of nitrogen in marine habitats, yet few N 2 -fixing cyanobacteria are found in the oligotrophic open ocean (Howarth et al, 1988;Michaels et al, 1996;Karl et al, 2002;Stal and Zehr, 2008). The free-living bloom-forming filamentous cyanobacterium Trichodesmium (Capone et al, 1997;LaRoche and Breitbarth, 2005) and the heterocystous Richelia symbionts of marine pelagic diatoms (Villareal, 1992;Foster and Zehr, 2006) were believed to be the primary N 2 -fixers (diazotrophs) until the discovery that the unicellular N 2 -fixing cyanobacteria were also generally widespread and important in N 2 -fixation in the open ocean (Zehr et al, 2001;Falcón et al, 2002;Mazard et al, 2004;Montoya et al, 2004;Langlois et al, 2005). One of these unicellular cyanobacterial species, Crocosphaera watsonii strain WH 8501 (C. watsonii), had previously been isolated from tropical waters (Waterbury and Rippka, 1989).…”

Significant N2 fixation by heterotrophs, photoheterotrophs and heterocystous cyanobacteria in two temperate estuaries

“…This implies that heterocystous cyanobacteria are not always a pre-requisite for pelagic N 2 fixation in temperate estuaries as previously assumed (Howarth et al, 1988b) and that significant N 2 fixation takes place in these environments. In both estuaries, the N 2 fixation was accompanied by diverse diazotrophic bacteria, mainly related to (photo)heterotrophic bacteria, including the supposedly tropical to subtropical group of unicellular cyanobacteria, UCYN-A (Mazard et al, 2004;Moisander et al, 2010), which has recently been shown to be a metabolically reduced symbiont of a unicellular prymnesiophyte (Thompson et al, 2012). Interestingly, nifH transcripts related to this group have been reported from Chesapeake Bay at water temperatures as low as 12 1C (Short and Zehr, 2007), yet, here we measured nifH transcript abundances of 410 4 l À 1 in RF in February at only 2.5 1C.…”